Agglomerated microbiological media

ABSTRACT

A method of making a flowable, dried agglomerated nutrient medium is provided. The method comprises introducing a nutrient component comprising a powdered nutrient, and an agglomeration liquid, into an agglomerator comprising a flow-through-type agglomeration chamber, wet-massing the nutrient component with the agglomeration liquid in the agglomeration chamber for a predetermined period of time to form agglomerated nutrient medium particles, and exposing the agglomerated nutrient medium particles to drying conditions for a period of time to form the dried, agglomerated nutrient medium. The nutrient component facilitates the growth of a microorganism. Compositions, articles, and kits comprising the flowable, dried agglomerated nutrient medium are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/415,744, filed Jan. 20, 2015, which is a 371 of International PatentApplication No. PCT/US2013/051449, filed Jul. 22, 2013, which claims thebenefit of U.S. Provisional Patent Application No. 61/675,624, filedJul. 25, 2012, which applications are incorporated herein by referencein their entirety.

BACKGROUND

Nutrient media formulations have been used to cultivate a number of celltypes including animal, plant and microbial cells. Cells cultivated inculture media catabolize available nutrients and produce usefulbiological substances such as monoclonal antibodies, hormones, growthfactors, viruses and the like. Such products have therapeuticapplications and, with the advent of recombinant DNA technology, cellscan be engineered to produce large quantities of these products. Thus,the ability to cultivate cells in vitro is not only important for thestudy of cell physiology, but is also necessary for the production ofuseful substances which may not otherwise be obtained by cost-effectivemeans.

Typical components of microbial culture media may include proteinhydrolysates, inorganic salts, vitamins, trace metals, andcarbohydrates, the types and amounts of which may vary depending uponthe particular requirements of a given species of microorganism. Becausethese components tend to be more stable in a dehydrated form, they arefrequently provided as dry, powdered formulations. The powderedformulations are added to water and, optionally, sterilized before use.

Culture media are typically produced in liquid form or in powdered form.Each of these forms has particular advantages and disadvantages.

For example, liquid culture medium has the advantage that it is providedready-to-use (unless supplementation with nutrients or other componentsis necessary), and that the formulations have been optimized forparticular cell types. Liquid media have the disadvantages, however,that they may require the addition of supplements (e.g., a vitamin orcofactor, and antibiotic) for optimal performance in cultivating aparticular microorganism. Furthermore, most liquid media require sometype of sterilization (e.g., autoclaving, filtration), which can be atime-consuming and/or expensive process.

To overcome some of the disadvantages, liquid culture medium can be madein concentrated form; the concentrate may be diluted to workingconcentrations prior to use. This approach provides the capability ofmaking larger and variable batch sizes than with standard culture media,and the concentrated media formulations or components thereof often havelonger shelf-life. U.S. Pat. No. 5,474,931 is directed to culture mediaconcentrate technology and is incorporated herein by reference in itsentirety. Despite these advantages, however, concentrated liquid mediastill have the disadvantages of their need for the addition ofsupplements, and may be difficult to sterilize economically.

As an alternative to liquid media, powdered culture media are oftenused. This approach has the advantages that larger batch sizes may beproduced, the powdered media typically have longer shelf lives thanliquid media, and the media can be sterilized by irradiation (e.g.,gamma or ultraviolet irradiation) or ethylene oxide permeation afterformulation. However, powdered media have several distinctdisadvantages. For example, some of the components of powdered mediabecome insoluble or aggregate upon lyophilization such thatresolubilization is difficult or impossible. Furthermore, powdered mediatypically comprise fine dust particles which can make them particularlydifficult to transfer and/or reconstitute without some loss of material,and which may further make them impractical for use in manybiotechnology production facilities operating under GMP/GLP, USP or ISO9000 settings.

Despite the advancements in rehydratable media, there still exists aneed for rapidly dissolving nutritionally complex stable dry powdernutritive media, media supplements, media subgroups and buffers, whichcan be prepared in variable bulk quantities and which are amenable tosterilization.

SUMMARY

The present disclosure generally relates to nutrient media that are usedto facilitate growth of a microorganism (e.g., a bacterium, a yeast, amold). In particular, the present disclosure provides methods ofagglomerating one or more powdered nutrient component to produce adried, agglomerated nutrient medium. The methods include processconditions that provide agglomerated particles having a selectedchemical composition, size, density, water content, or a combination ofany two or more of the foregoing properties. In another aspect, thepresent disclosure further provides a composition comprising a driedagglomerated nutrient medium made according to any method disclosedherein. In another aspect, the present disclosure provides an articlesand kits comprising any dried agglomerated nutrient compositiondisclosed herein.

Advantageously, the compositions of the present disclosure have achemical composition that facilitates wetting of the surface of theparticles by an aqueous solvent. Further advantageously, thecompositions of the present disclosure have a density (e.g., agravity-packed mean particle density) selected to facilitate submersionof the particles in an aqueous solvent. Even further advantageously, adried agglomerated nutrient medium of the present disclosure has aparticle size distribution selected to facilitate rapid dissolution ofthe particles in an aqueous solvent.

In one aspect, the present disclosure provides a method of making aflowable, dried agglomerated nutrient medium. The method can compriseintroducing a nutrient component comprising a powdered nutrient, and anagglomeration liquid, into an agglomerator comprising aflow-through-type agglomeration chamber; wet-massing the nutrientcomponent with the agglomeration liquid in the agglomeration chamber fora predetermined period of time to form agglomerated nutrient mediumparticles; and exposing the agglomerated nutrient medium particles todrying conditions for a period of time to form the dried, agglomeratednutrient medium. The nutrient component facilitates the growth of amicroorganism. The agglomeration liquid is introduced as an atomizedspray. In any embodiment of the method, the agglomeration liquid canconsist of water. In any embodiment of the method, the agglomerationliquid can comprise a solvent having a dissolved nutrient thatfacilitates the growth of a microorganism. In any of the aboveembodiments of the method, introducing a nutrient component can compriseintroducing a substantially uniform mixture of two or more powderednutrients.

In any of the above embodiments of the method, the agglomerator furthercan comprise a mixing blade, wherein introducing a nutrient componentinto the agglomeration chamber comprises introducing the nutrientcomponent at a feed rate of about 50 kg/hour to about 1000 kg/hour;wherein introducing an agglomeration liquid into the agglomerationchamber comprises introducing the agglomeration liquid at a spray rateof about 15 kg/hour to about 300 kg/hour; wherein the mixing blade isrotated at a blade rotation rate of about 500 rpm to about 4000 rpm;wherein exposing the agglomerated nutrient medium to drying conditionscomprises exposing the agglomerated nutrient medium to a predeterminedtemperature of about 60° C. to about 85° C.; and wherein the feed rate,the spray rate, the blade rotation rate, and the predeterminedtemperature are selected to produce a dried agglomerated nutrient mediumhaving a preselected mean water content of less than 5 weight percent.In any of the above embodiments of the method, the agglomerator furthercan comprise a mixing blade, wherein introducing a nutrient componentinto the agglomeration chamber comprises introducing the nutrientcomponent at a feed rate of about 50 kg/hour to about 1000 kg/hour;wherein introducing an agglomeration liquid into the agglomerationchamber comprises introducing the agglomeration liquid at a spray rateof about 15 kg/hour to about 300 kg/hour; wherein the mixing blade isrotated at a blade rotation rate of about 500 rpm to about 4000 rpm;wherein exposing the agglomerated nutrient medium to drying conditionscomprises exposing the agglomerated nutrient medium to a predeterminedtemperature of about 60° C. to about 85° C.; and wherein the feed rate,the spray rate, the blade rotation rate, and the predeterminedtemperature are selected to produce a dried agglomerated nutrient mediumhaving a gravity-packed density of 0.2 to 0.5 grams per cubiccentimeter. In any of the above embodiments of the method, theagglomerator further can comprise a mixing blade, wherein introducing anutrient component into the agglomeration chamber comprises introducingthe nutrient component at a feed rate of about 50 kg/hour to about 1000kg/hour; wherein introducing an agglomeration liquid into theagglomeration chamber comprises introducing the agglomeration liquid ata spray rate of about 15 kg/hour to about 300 kg/hour; wherein themixing blade is rotated at a blade rotation rate of about 500 rpm toabout 4000 rpm; wherein exposing the agglomerated nutrient medium todrying conditions comprises exposing the agglomerated nutrient medium toa predetermined temperature of about 60° C. to about 85° C.; and whereinthe feed rate, the spray rate, the blade rotation rate, and thepredetermined temperature are selected to produce a dried agglomeratednutrient medium having particles of a first particle size distributionrange of about 105 microns to about 2000 microns. In any of the aboveembodiments, the method further can comprise selecting a subpopulationof the particles, the subpopulation having a second particle sizedistribution range that is narrower than the first particle sizedistribution range, the second particle size distribution range beingabout 149 microns to about 1000 microns.

In any of the above embodiments of the method, one or more powderednutrient can be selected from the group consisting of a protein, acarbohydrate, a salt and a mixture of any two or more of the foregoingpowdered nutrients. In any of the above embodiments, the method furthercan comprise subjecting the dried agglomerated nutrient medium to aprocess that reduces the number of viable microorganisms in the driedagglomerated nutrient medium.

In another aspect, the present disclosure provides a compositioncomprising the flowable, dried agglomerated nutrient medium produced bythe method of any one of the preceding claims. In any embodiment of thecomposition, a specified mass of the dried agglomerated nutrient mediumcan dissolve more rapidly than an equal mass of the nutrient componentfrom which the agglomerated nutrient medium was made.

In yet another aspect, the present disclosure provides a kit comprisingany of the above embodiments of the composition. In any embodiment ofthe kit, the composition can be disposed in a package comprising apredetermined mass of the dried agglomerated nutrient medium, whereinthe predetermined mass can be mixed with 90 mL to 99 mL of aqueousdiluent to produce a reconstituted medium capable of facilitating thegrowth of a microorganism. In any of the above embodiments, the kitfurther can comprise an article selected from the group consisting of abag, a bottle, and a sample acquisition device. In any of the aboveembodiments, the kit further can comprise a selective agent and/or anindicator reagent.

In yet another aspect, the present disclosure provides a thin filmculture device. The culture device can comprise a self-supportingwaterproof substrate having first and second major surfaces; an adhesivelayer disposed on at least a portion of the first major surface; acoating comprising the composition of any of the above embodimentsdisposed on a least a portion of the adhesive layer; and a dry,cold-water soluble gelling agent positioned for fluidic contact with thenutrient medium. In any of the above embodiments, the thin film culturedevice further can comprise a cover sheet coupled to a least a portionof the substrate, the cover sheet having a first side facing the firstmajor surface.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably. Thus, for example, “a” particle can be interpretedto mean “one or more” particles.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

Additional details of these and other embodiments are set forth in theaccompanying drawings and the description below. Other features, objectsand advantages will become apparent from the description and drawings,and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a top perspective view, partially in section, of oneembodiment of a thin film culture device according to the presentdisclosure.

DETAILED DESCRIPTION

Before any embodiments of the present disclosure are explained indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thefollowing drawings. The invention is capable of other embodiments and ofbeing practiced or of being carried out in various ways. Also, it is tobe understood that the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having” and variations thereofherein is meant to encompass the items listed thereafter and equivalentsthereof as well as additional items. It is to be understood that otherembodiments may be utilized and structural or logical changes may bemade without departing from the scope of the present disclosure.

In general, the present disclosure relates to flowable, driedagglomerated particles made from water-soluble powders and a method ofmaking said agglomerates. In some embodiments, the method can be used tomake a nutrient medium for facilitating the growth of a microorganism.In contrast with at least one of the powders from which the agglomeratedparticles are made, and in contrast with other agglomerated particlecompositions, a population of the inventive agglomerated particlesadvantageously possess two highly-desirable properties: 1) theagglomerated particles rapidly (i.e., immediately or almost immediately)and substantially quantitatively sink below the surface of an aqueousliquid when poured onto the surface of the aqueous liquid and 2) theagglomerated particles rapidly dissolve into an aqueous liquid. Withoutbeing bound by theory, it is believed these highly-desirable propertiesresult from the average size, surface area, surface composition, densityand/or a unique combination of two or more of the foregoing physicalattributes found in populations of agglomerated particles that areproduced according to the present disclosure. Thus, in one aspect, thepresent disclosure provides a nutrient medium that rapidly disperses anddissolves into an aqueous liquid with little or no manual or mechanicalagitation.

The present disclosure thus provides a method for the preparation of anagglomerated nutrient medium, the agglomerated medium being suitable tofacilitate the recovery and/or growth of a microorganism, the methodcomprising i) introducing a nutrient component comprising a powderednutrient and an agglomeration liquid into a flow-through-typeagglomeration chamber (i.e., a “Schugi-type” agglomeration chamber), ii)wet-massing the nutrient component with the agglomeration liquid in theagglomeration chamber for a period of at the most 30 seconds to form anagglomerated nutrient medium, and iii) exposing the agglomeratednutrient medium to drying conditions for a period of time. According tothe method of the present disclosure, the nutrient component (e.g., atleast one powdered nutrient) facilitates the growth of a microorganism.Further, according to the method of the present disclosure, theagglomeration liquid is introduced as an atomized spray.

It has been found that a very short and intensive step of application ofthe agglomeration liquid and wet-massing of the composition comprisingthe nutrient leads to a population of agglomerated particles that issuitable for the preparation of a flowable dried nutrient medium.

The process time for the step of the application of the agglomerationliquid and wet-massing may vary depending on the equipment employed. Ina specific embodiment, the wet-massing in step ii) is carried out in atime period of at the most about 20 sec such as, e.g., at the most about15 sec, at the most about 10 sec or at the most about 5 sec.

An especially suitable apparatus for use in the present method is avertical-path, flow-through-type agglomeration chamber manufactured byBEPEX International LLC. The model FX-100 Schugi FLEX-O-MIXmixer/agglomerator is preferred for the methods of the presentdisclosure, but the invention is not limited to the use of thisparticular model or manufacturer, other models and apparatus having asimilar construction leading to a very fast step of wet-massing asmentioned above, are also within the scope of the present disclosure. Ine.g. the Schugi FLEXOMIX, the wet-massing in step ii) is carried out ina time period of at the most about 1 sec preferably at the most about0.5 sec, at the most about 0.4 sec, at the most about 0.3 sec or at themost about 0.2 sec.

A person having ordinary skill in the art will recognize, depending onthe size of the apparatus employed, the feeding rate of theagglomeration chamber with the nutrient powder composition as well asthe flow rate of the agglomeration liquid may vary.

Normally, in flow-through-type agglomeration apparatuses such as thoseused in according to the present disclosure, the agglomeration chamberis fed with the nutrient component (e.g. a powdered nutrient) using afeed rate from about 50 to about 1000 kg/h such as, e.g., from 50 toabout 850 kg/h, from about 50 to about 750 kg/h, from 50 to about 500kg/h, from about 50 to about 250 kg/h, from about 50 to about 200 kg/h,from about 100 to about 200 kg/h such as about 100 kg/h or about 200kg/h.

In flow-through-type agglomeration equipment intended for very fastproduction, the agglomeration chamber is fed with a feeding rate ofabout 1000 to about 1500 kg/h such as, e.g., about 1100 to about 1300kg/h.

The liquid flow of the agglomeration liquid is also an importantparameter in a flow-through-type agglomeration chamber in order toobtain a fast and efficient step of application of the agglomerationliquid and wet-massing the composition. Normally, the agglomerationliquid is sprayed on the composition at a spray rate in a range of fromabout 15 to about 100 kg/h. Again, the flow rate depends on the size(e.g., chamber volume) of the equipment; larger equipment enables ahigher flow rate than smaller equipment. In the exemplary apparatusdescribed above (model FX-100 Schugi FLEX-O-MIX mixer/agglomerator), theagglomeration liquid can be sprayed on the composition at a spray ratein a range from about 15 to about 80 kg/h, inclusive; such as, e.g.,from about 20 to about 60 kg/h, inclusive; from about 20 to about 50kg/h, inclusive; from about 20 to about 40 kg/h, inclusive; or fromabout 25 to about 35 kg/h, inclusive. A person skilled in the art willknow how to adjust the liquid flow depending on the equipment employed.As a guidance for larger equipment than that employed in the examples,the agglomeration liquid may be sprayed on the composition at a sprayrate in a range from about 50 to about 300 kg/h, inclusive; such as,e.g., from about 60 to about 200 kg/h, inclusive; from about 65 to about150 kg/h, inclusive; from about 70 to about 125 kg/h, inclusive; or fromabout 75 to about 105 kg/h, inclusive.

In any embodiment, the nutrient component and agglomeration liquid canbe fed into the agglomeration chamber simultaneously and/orcontinuously. The continuous feeding of the powder composition as wellas of the agglomeration liquid enables the process to be continuous,i.e. it may be continuously for a period of 1 day or more such as, e.g.,2 days or more, a 3 days or more, 4 days or more, 5 days or more or 7days or more. In principle, the time period is determined based on theamount of dried agglomerated medium to be produced, but it mayeventually be interrupted by clogging of parts of the apparatus thatneed to be rinsed.

In a specific embodiment using the Schugi FLEXOMIX equipment (or asimilar apparatus) the nutrient component comprising the powderednutrient (e.g., the powdered ingredients to make buffered peptone water)is fed through the top of the agglomeration chamber and passes throughthe chamber by means of gravity. The chamber contains a number of bladesthat enable an intensive wet-massing of the nutrient component. Comparedwith a high-shear mixing process, the step of application of theagglomeration liquid and the wet-massing according to the present methodis much faster.

Nutrient Component

Methods of the present disclosure comprise a step of wet-massing anutrient component with an agglomeration liquid. The nutrient componentcomprises at least one nutrient that facilitates the growth of amicroorganism (e.g., a bacterium, a yeast, a mold). In any embodiment,the nutrient component may comprise a mixture of two or more nutrientsthat facilitate the growth of a microorganism. Non-limiting examples ofsuitable nutrients include a carbohydrate (e.g., a monosaccharide, adisaccharide, a trisaccharide, an oligosaccharide, or a polysaccharide),a protein, a protein hydrolysate, a cellular extract (e.g., yeastextract), a salt, a buffer component, a selective agent (e.g., anantibiotic), and a combination of any two or more of the foregoingnutrients.

In any embodiment, the nutrient component can be a substantially dryformulation (e.g., a substantially dry powder). Powdered media aretypically produced by admixing the dried components of the culturemedium via a mixing process, e.g., ball-milling, or by lyophilizingpremade liquid culture medium. An exemplary nutrient medium that is usedin the method of the present disclosure is buffered peptone water, whichis commercially-available as a powder from a variety of sourcesincluding, for example, Oxoid Limited (Hampshire, UK) and Sigma-Aldrich(St. Louis, Mo.).

Other powdered nutrient media formulations that can be agglomeratedaccording to the present disclosure include formulations that arerehydrated to produce buffered peptone water, trypticase soy broth,lactose broth, UVM modified Listeria enrichment broth, Buffered ListeriaEnrichment broth ((“BLEB”), Demi-Fraser broth, as well as other culturemedium for microorganisms that are known in the art and may be found,for example in the Handbook of Microbiological Media (R. Atlas, ed.,4^(th) Edition, 2010, CRC Press, Ann Arbor, Mich.), which isincorporated herein by reference in its entirety.

Agglomeration Liquid

An agglomeration liquid is sprayed into the agglomeration chamber topromote agglomeration of the smaller individual particles (e.g., powderparticles) of the nutrient component into larger agglomerated particles.In any embodiment, the agglomeration liquid can comprise water (e.g.,distilled and/or deionized water). In any embodiment, the agglomerationliquid can consist essentially of water (e.g., distilled and/ordeionized water). In any embodiment, the agglomeration liquid canconsist of water (e.g., distilled and/or deionized water). In anyembodiment, the agglomeration liquid may comprise an organic solvent(e.g., ethanol, isopropanol).

Optionally, in any embodiment, a binder, especially a water-solublebinder, may be useful to facilitate the agglomeration of the nutrientcomponent. The binder may be added (e.g., in the form of substantiallydry particles such as a powder, for example) to the nutrient componentor it may be dissolved or suspended in the agglomeration liquid.

Nonlimiting examples of suitable binders include biocompatible polymers(e.g. proteins, polyethylene glycol, polyvinylpyrollidone, polyvinylalcohol, polysaccharides, dextrans, dextrins, maltodextrins,microcrystalline cellulose, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, starch, andbiocompatible derivatives of any of the foregoing polymers) and sugars(e.g., dextrose, fructose, glucose, inositol, erythritol, isomalt,lactitol, lactose, maltitol, maltose, mannitol, sorbitol, sucrose,tagatose, trehalose, and xylitol). In any embodiment, the binder may bea substance that facilitates the growth of a microorganism (e.g., acomplete medium or diluent containing at least one nutrient). By way ofexample, a suitable agglomeration liquid for agglomerated bufferedpeptone water medium can be an solution of 1-50 weight percent bufferedpeptone water and a suitable agglomeration liquid for agglomeratedtrypticase soy broth medium cab be an aqueous solution of 1-50 weightpercent trypticase soy medium.

The concentration of the binder in the composition comprising thenutrient may vary over a great range depending on the particular binderemployed, but in general it is between from about 0.1% to about 40% w/wsuch as, e.g. from about 0.2 to about 35% w/w, from about 0.3 to about30% w/w or from about 0.4 to about 25% w/w or from about 0.4 to about24.2% w/w. In the case of sorbitol, the concentration is normally about20-30% w/w and in the case of sugar alcohols other than sorbitol; theconcentration is normally in the higher range such as from about 30 toabout 40% w/w.

Preferably, the agglomeration liquid is an aqueous medium. In the casewhere the binder is included in the agglomeration liquid, theagglomeration liquid is prepared by dissolving the binder in water.Alternatively the binder can be admixed in a dry form to the powder.

The agglomerated nutrient medium is subjected to drying in a suitabledrying chamber. It maybe a drying chamber that is operatively coupled tothe agglomeration apparatus. The drying chamber may be operated batchwise or continuously and, optionally, may be segregated into two or moreindependently-controlled drying zones, the zones being operated atdifferent temperatures.

In any embodiment, the agglomeration liquid may comprise a nutrientmedium that facilitates the growth of a microorganism. For example, inone embodiment, the agglomeration liquid may comprise buffered peptonewater broth (water, 10.0 g/L peptone, 5.0 g/L sodium chloride, 9.0 g/Ldisodium hydrogen orthophosphate 12H₂O, and 1.5 g/L potassium dihydrogenorthophosphate).

A method according to the present disclosure is used to make anagglomerated nutrient medium (e.g., a dried, wet-agglomerated nutrientmedium). The agglomerated nutrient medium may be an agglomeratedmicrobiological nutrient medium that facilitates the growth of amicroorganism (e.g., a bacterium, a yeast, a mold). A method of thepresent disclosure comprises introducing a nutrient component comprisinga powdered nutrient, and an agglomeration liquid, into an agglomerationchamber. The agglomeration chamber can be a component of a system thatuses the Schugi mixing method (see, for example, “The Art ofAgglomeration”, 1999, Powder Handling & Processing, volume 11, page 368,which is incorporated herein by reference in its entirety) to produceagglomerated particles. An exemplary system is the vertical path SchugiFLEXOMIX FXD-100 mixer/agglomerator, available from Bepex International,LLC (Minneapolis, Minn.). The agglomeration chamber can have a firstend, a second end opposite the first end and a mixing blade (e.g., aknife blade) disposed therebetween. The chamber may be configured with asubstantially vertical path for the passage of materials from the firstend of the chamber to the second end of the chamber.

In some embodiments, the mixing blade forms an angle with the shaft towhich it is attached. The blade angle can be adjusted between 75° and105° relative to the shaft. A person having ordinary skill in the artwill recognize the blade angle can be adjusted to achieve optimalcontrol of the particle size and particle size distribution of theagglomerated nutrient medium particles.

In any embodiment, the nutrient component and the agglomeration fluidcan be introduced into the agglomeration chamber proximate the first endof the chamber. In any embodiment, the nutrient component can be fed(e.g., by gravity flow) into the agglomeration chamber via a powderdispenser. In any embodiment, the agglomeration fluid may be introducedinto the chamber via one or more nozzle that forms an atomized spray(e.g., an air-driven atomized spray). The nutrient component can be anysuitable nutrient component described herein. In any embodiment, thenutrient component can comprise a substantially uniform mixture of twoor more powdered nutrients.

In any embodiment, a method of the present disclosure further compriseswet-massing the nutrient component with the agglomeration liquid in theagglomeration chamber for a predetermined period of time (e.g., at themost, about 30 seconds; at the most, about 20 seconds; at the most,about 10 seconds; at the most, about 5 seconds; at the most, about 1second; preferably at the most, about 0.5 sec; at the most, about 0.4seconds; at the most, about 0.3 seconds; or at the most, about 0.2seconds) to form an agglomerated nutrient medium. In any embodiment, thepredetermined period of time can be selected to result in agglomeratedparticles with a particular water content or a particular range of watercontents. In any embodiment, a mixing blade (e.g., a rotating knifeblade) can be used to facilitate contact between the nutrient componentand the agglomerator fluid and/or to limit the size of the agglomeratednutrient medium particles.

In any embodiment, the method further comprises exposing theagglomerated nutrient medium particles to drying conditions for a periodof time. In any embodiment, the agglomerated nutrient medium particlescan be exposed to a gas (e.g., air), preferably at ambient temperatureor higher, to cause drying of the particles. In any embodiment, thedrying conditions (e.g., temperature, time, and gas or air flow) can beselected to produce agglomerated nutrient medium particles having apreselected average water content and/or a preselected range of watercontent. In any embodiment, the drying temperature can be about 60° C.to about 85° C., inclusive. In any embodiment, the drying temperaturecan be about 60° C. to about 65° C., inclusive. In any embodiment, thedrying temperature can be about 65° C. to about 70° C., inclusive. Inany embodiment, the drying temperature can be about 70° C. to about 75°C., inclusive. In any embodiment, the drying temperature can be about75° C. to about 80° C., inclusive. In any embodiment, the dryingtemperature can be about 80° C. to about 85° C., inclusive.

In some embodiments, the mixing chamber may be operatively coupled to adryer (e.g., a fluidized bed dryer) such that, after the agglomeratednutrient medium particles pass (e.g., by gravity flow) from the firstend to the second end of the mixing chamber the particles move into adrying zone (e.g., the fluidized bed dryer). In these embodiments, thetemperature, gas (e.g., air) flow, and residence time in the drying zonecan be preselected to yield particles with a preselected water content.In any embodiments, the dryer may comprise a plurality of segregateddrying zones, each zone having a particular temperature and residencetime parameter for processing the particles. In any embodiment, thedrier may comprise a cooling zone.

In any embodiment of the method, the agglomeration liquid may compriseat least one dissolved nutrient that facilitates the growth of amicroorganism. In one embodiment, the agglomeration liquid comprisesbuffered peptone water.

A method according to the present disclosure comprises introducing anutrient component into a agglomeration chamber. In any embodiment,introducing a nutrient component into the agglomeration chambercomprises introducing the nutrient component at a feed rate. A personhaving ordinary skill in the art will recognize the rates of introducingthe nutrient component and the agglomeration liquid can affect thephysical parameters (e.g., size, mass, density, and shape) of theagglomerated particles. Furthermore, the feed rate typically will varyaccording to the volume and geometry of the agglomeration chamber. Thus,in an embodiment of the method wherein the agglomeration process isconducted in a Schugi FLEXOMIX FX-100 agglomerator available fromHosokawa Micron (Summit, N.J.), the feed rate of the nutrient componentcan be from about 200 to about 1000 kg/h (e.g., from 300 to about 850kg/h, from about 300 to about 750 kg/h, from 300 to about 700 kg/h, fromabout 350 to about 650 kg/h, from about 400 to about 600 kg/h, fromabout 450 to about 550 kg/h, such as about 500 kg/h.

A method according to the present disclosure comprises introducing anagglomeration liquid into a agglomeration chamber. In any embodiment,introducing an agglomeration liquid into the agglomeration chambercomprises introducing the agglomeration liquid at a spray rate. A personhaving ordinary skill in the art will recognize the rates of introducingthe nutrient component and the agglomeration liquid can affect thephysical parameters (e.g., size, mass, density, and shape) of theagglomerated particles. Furthermore, the spray rate typically will varyaccording to the volume and geometry of the agglomeration chamber andthe feed rate and composition of the powder. Thus, in an embodiment ofthe method wherein the agglomeration process is conducted in a SchugiFLEXOMIX FX-100 agglomerator available from Hosokawa Micron (Summit,N.J.), the spray rate of the agglomeration liquid can be in a range fromabout 15 to about 80 kg/h, inclusive; such as, e.g., from about 20 toabout 60 kg/h, inclusive; from about 20 to about 50 kg/h, inclusive;from about 20 to about 40 kg/h, inclusive; or from about 25 to about 35kg/h, inclusive. A person skilled in the art will know how to adjust theliquid flow depending on the equipment employed. As a guidance forequipment that is larger than a Schugi FLEXOMIX FX-100 agglomerator, theagglomeration liquid may be sprayed into the agglomeration chamber at aspray rate in a range from about 50 to about 300 kg/h, inclusive (e.g.,from about 60 to about 200 kg/h, inclusive; from about 65 to about 150kg/h, inclusive; from about 70 to about 125 kg/h, inclusive; or fromabout 75 to about 105 kg/h, inclusive).

In any embodiment, the agglomerator used in a method of the presentdisclosure can comprise a mixing blade. The mixing blade may be coupledto a shaft. The mixing blade is rotated during the agglomeration processto accomplish one or more functions including, but not limited to i) tofacilitate contact between the nutrient component and the agglomerationliquid, ii) to control the particle size of the agglomerated particlesand/or iii) to facilitate the movement of particles through theagglomeration chamber. In any embodiment, the mixing blade can berotated at about 500 rpm to about 5000 rpm, inclusively. In someembodiments, the mixing blade can be rotated at 3000 rpm. In someembodiments, the mixing blade can be rotated at 4000 rpm. A personhaving ordinary skill in the art will recognize the rotation rate and/orgeometry of the mixing blade may be used to control the particle shape,effective particle size (e.g., average particle size and/or particlesize range), density, and/or packed density of the agglomerated nutrientmedium particles, as described above.

Thus, at least some of the process conditions (e.g., nutrient feed rate,agglomeration liquid spray rate, blade rotation rate, and/orpredetermined drying temperature) can affect one or more of thephysicochemical properties of the dried agglomerated nutrient mediumparticles produced by the method if the present disclosure. Theseproperties include, but are not limited to the average effectiveparticle diameter, the average particle diameter, the particle shape,the average particle mass, the average particle moisture content, therange of effective particle diameters, the range of particle masses,and/or the range of particle moisture content. One or a combination ofany two or more of the physicochemical properties can affect thehydrodynamic properties (e.g., particle sink rate in an aqueous liquid,particle dissolution time in an aqueous liquid) of the dried,agglomerated nutrient medium particles.

In any embodiment, a method according to the present disclosure yields apopulation of dried, agglomerated nutrient medium particles having anaverage effective particle diameter and a first particle sizedistribution range. In any embodiment, the first particle sizedistribution range can comprise agglomerated particles having aneffective particle diameter from about 105 microns to about 2000microns. In any embodiment, the first particle size distribution rangecan comprise agglomerated particles having an effective particlediameter from about 105 microns to about 1000 microns.

In any embodiment, the method further can comprise isolating asubpopulation of the dried, agglomerated nutrient medium particles, thesubpopulation having a preselected average effective particle diameterand/or having an effective particle diameter that falls within a secondparticle size distribution range. The subpopulation can be isolatedusing particle size-selection methods known in the art (e.g., usingsieves that permit the retention or passage of particles withpredetermined effective particle diameters. In any embodiment, thesubpopulation may include agglomerated particles having an effectiveparticle diameter from about 149 microns to about 1000 micron,inclusive. In any embodiment, the subpopulation may include agglomeratedparticles having an effective particle diameter from about 149 micronsto about 850 microns, inclusive. In any embodiment, the subpopulationmay include agglomerated particles having an effective particle diameterfrom about 149 microns to about 400 microns, inclusive. In anyembodiment, the subpopulation may include agglomerated particles havingan effective particle diameter from about 250 microns to about 400microns, inclusive.

In any embodiment of the method, the dried agglomerated nutrient mediumparticles can have a preselected mean water content. A person havingordinary skill in the art will recognize the desired mean water contentcan be achieved by adjusting certain process parameters such as, forexample, the spray rate, the drying temperature, and/or the drying time.The mean water content can be measured, for example, using the KarlFischer titration method. In any embodiment, the mean weight percentwater can be preselected to be less than about 5 weight percent water.In any embodiment, the mean weight percent water can be preselected tobe less than or equal to about 3 weight percent water.

In any embodiment of the method, the dried agglomerated nutrient mediumparticles can have a gravity-packed density. The gravity-packed densityof the dried, agglomerated nutrient medium particles can be measured,for example, by pouring a predefined volume (e.g., 100 mL) of theparticles into a graduated cylinder, and measuring the mass of theparticles in order to calculate the average density. The gravity-packeddensity can be from about 0.2 grams per cubic centimeter to about 0.5grams per cubic centimeter.

In any of the above embodiments, a method of the present disclosurefurther can comprise subjecting the dried agglomerated nutrient mediumto a process that reduces the number of viable microorganisms in thedried agglomerated nutrient medium. In any embodiment, the driedagglomerated nutrient medium can be treated with ethylene oxide vapor inorder to reduce the number of viable microorganisms according to methodsthat are known in the art. After treatment with ethylene oxide vapor,the nutrient medium can be allowed to aerate before use, in order toreduce or eliminate residual ethylene oxide in the ethyleneoxide-treated agglomerated medium. In any embodiment, the driedagglomerated nutrient medium can be treated with ionizing radiation(e.g., gamma radiation) or a source of ultraviolet light in order toreduce the number of viable microorganisms according to methods that areknown in the art. The dried, agglomerated medium can be exposed to asource of gamma irradiation as described in U.S. Pat. No. 6,383,810,which is incorporated herein by reference in its entirety.

In addition to methods of making a flowable, dried agglomerated nutrientmedium, the present disclosure further provides a composition offlowable, dried agglomerated nutrient medium prepared according to anyof the embodiments of the method disclosed herein. The composition canbe used to prepare nutrient medium to facilitate the recovery and/orgrowth of a microorganism in a sample (e.g., a clinical sample, anenvironmental sample, a food sample, a beverage sample, a water sample).Moreover, the composition can be used in an article for culturingmicroorganisms.

Advantageously, a composition of the present disclosure is flowable(e.g., can be poured from one container into another), is substantiallyfree of dust (e.g., the smaller particles of nutrient from which theagglomerated medium is made and, when the agglomerated medium is madefrom a substantially dry starting material that includes at least onewater-soluble powder, the agglomerated medium can dissolve in watersubstantially faster than the starting material. When the dissolutiontime is measured by pouring 2.55 g to 4.44 grams of a dried,agglomerated nutrient medium (e.g., a dried, agglomerated nutrientmedium used to make buffered peptone water, trypticase soy broth, ormodified Listeria recovery medium, for example) into a vessel containing100 mL of deionized water and holding the vessel at room temperaturewithout stirring, the dried agglomerated medium can dissolve in lessthan 5 minutes. In some embodiments, the dried agglomerated medium candissolve in less than 4 minutes. In some embodiments, the driedagglomerated medium can dissolve in less than 3 minutes. In someembodiments, the dried agglomerated medium can dissolve in less than 2minutes. In some embodiments, the dried agglomerated medium can dissolvein less than 1 minute.

Thus, in yet another aspect, the present disclosure provides an articlefor culturing a microorganism. A nonlimiting example of an articleaccording to the present disclosure is a thin film culture devicesimilar to the powder-coated devices described in U.S. Pat. Nos.4,565,783 and 5,089,413; which are incorporated herein by reference intheir entirety. The dried, agglomerated nutrient medium of the presentdisclosure can be used in place of the powdered nutrients in the thinfilm culture devices.

FIG. 1 shows one embodiment of a thin film culture device according tothe present disclosure. The culture device 10 includes a body membercomprising a self-supporting water-proof substrate 12 having upper andlower surfaces. Substrate 12 is preferably a relatively stiff film of amaterial such as polyester, polypropylene or polystyrene which will notabsorb or otherwise be affected by water. Polyester films approximately0.004 to 0.007 inch (0.1 to 0.18 mm) thick, polypropylene filmsapproximately 0.004 to 0.008 inch (0.1 to 0.2 mm) thick and polystyrenefilms approximately 0.015 inch (0.38 mm) thick have been found to workwell. Other suitable substrates include paper with a polyethylene orother water-proof coating. An example of a suitable polyethylene-coatedpaper substrate is “Schoeller Type MIL” photoprint paper (commerciallyavailable from Schoeller Pulaski, New York, N.Y.). The substrate 12 maybe either transparent or opaque, depending on whether one wishes to viewbacterial colonies through the substrate. To facilitate the counting ofbacterial colonies, the substrate 12 preferably has a square gridpattern printed thereon as described in U.S. Pat. No. 4,565,783, forexample.

Substrate 12 is coated on its upper surface with a layer of an adhesive14 which serves to hold the dry gelling agent and/or nutrients in auniform monolayer for easy hydration. Adhesive 14 should bewater-insoluble and non-inhibitory to the growth of microorganisms.Preferably, the adhesive is sufficiently transparent when wet to enablethe viewing of bacterial colonies through the film coated with theadhesive. It is preferred that adhesive 14 be pressure-sensitive.However, heat-activated adhesives wherein a lower melting substance iscoated onto a higher melting substance may also be used. Water-activatedadhesives such as mucilage may also be useful.

Adhesive 14 should be coated onto substrate 12 in a thickness which ispreferably less than the diameter of the particles of the powderedgelling agent and/or nutrients. The object is to apply enough adhesiveto adhere the particles to the substrate but not so much that theparticles become completely embedded in the adhesive. A uniformmonolayer of powder 16 is desired with sufficient surface area exposedfor hydration. Generally, an adhesive layer in the thickness range of0.0002 to 0.0005 inch is suitable. The powders, including the dried,agglomerated nutrient medium of the present disclosure, can be coatedonto the adhesive layer as described in U.S. Pat. No. 4,565,783, forexample.

Adhered to one edge of spacer 18 of the body member is an optional coversheet 22. Cover sheet 22 is preferably transparent to facilitatecounting of the bacterial colonies and is substantially impermeable tobacteria and water vapor. As used in the specification and claims,“substantially impermeable to bacteria and moisture vapor” designatescover sheets which prevent undesired contamination of the dehydratedmedium during storage and use of the devices and which will provide anenvironment which will support the growth of microorganisms during theincubation period. Generally, it will have the same properties assubstrate 12, but need not be as stiff.

Although the illustrated embodiment of FIG. 1 includes a cover sheet 22attached to the device, it is also contemplated within the scope of theinvention that the powder-containing embodiments may be uncovered andsimply placed in a sterile environment during storage and incubation.Optionally, the cover sheet 22 further may comprise a layer of adhesive(not shown) and powder (not shown). The adhesive and powder adhered tothe cover sheet can have similar (or identical) properties and/orcomposition as the adhesive 14 and powder 16 described above and can beapplied to the cover sheet in the same manner as adhesive 14 and powder16 are applied to the substrate 12.

In another aspect, the present disclosure provides a kit. The kit cancomprise any composition comprising the dried, agglomerated nutrientmedium described herein. In any embodiment, the kit may includeinstructions for using the dried, agglomerated nutrient medium. In anyembodiment, the kit may further comprise an article for obtaining asample, processing a sample, and/or for culturing a microorganism. Thearticle can be selected from the group consisting of a bag, a bottle, asample acquisition device (e.g., a pipette, a swab, a sponge), and acombination of any two or more of the foregoing articles. In any of theabove embodiments, the kit further may comprise a selective agent (e.g.,an antibiotic). In any embodiment, the selective agent can be providedin a solution or in a substantially dry form. In any embodiment, theselective agent can be provided in a unit-dose form (e.g., a tablet,tube, or ampoule) that can be added to a predetermined volume of sample.In any embodiment, the kit further may comprise an indicator reagentused to detect a presence or an absence of a microorganism in a sample.

In any of the above embodiments, the kit may comprise a packagecomprising a predetermined mass of the dried agglomerated nutrientmedium. In any embodiment, the predetermined mass can be an amountsufficient to be mixed with a predetermined volume (e.g., 9.9 mL, 10 mL,90 mL, 99 mL, 100 mL, 225 mL, 1.0 L, or 3.375 L) of aqueous diluent toproduce a reconstituted medium capable of facilitating the growth of amicroorganism.

Certain embodiments of the methods, compositions, articles, and kits ofthe present disclosure are set forth in the following list ofembodiments.

EMBODIMENTS

Embodiment A is a method of making a flowable, dried agglomeratednutrient medium, the method comprising:

introducing a nutrient component comprising a powdered nutrient, and anagglomeration liquid, into an agglomerator comprising aflow-through-type agglomeration chamber;

-   -   wherein the nutrient component facilitates the growth of a        microorganism;    -   wherein the agglomeration liquid is introduced as an atomized        spray;

wet-massing the nutrient component with the agglomeration liquid in theagglomeration chamber for a predetermined period of time to formagglomerated nutrient medium particles; and

exposing the agglomerated nutrient medium particles to drying conditionsfor a period of time to form the dried, agglomerated nutrient medium.

Embodiment B is the method of Embodiment A, wherein the agglomerationliquid consists of water.

Embodiment C is the method of Embodiment A, wherein the agglomerationliquid comprises a solvent having a dissolved nutrient that facilitatesthe growth of a microorganism.

Embodiment D is the method of Embodiment C, wherein the solventcomprises water, wherein the nutrient comprises peptone, sodiumchloride, and sodium phosphate or potassium phosphate.

Embodiment E is the method of any one of the preceding Embodiments,wherein introducing a nutrient component comprises introducing asubstantially uniform mixture of two or more powdered nutrients.

Embodiment F is the method of any one of the preceding Embodiments:

wherein introducing a nutrient component into the agglomeration chambercomprises introducing the nutrient component at a feed rate of about 50kg/hour to about 1000 kg/hour;

wherein introducing a agglomeration liquid into the agglomerationchamber comprises introducing the agglomeration liquid at a spray rateof about 15 kg/hour to about 300 kg/hour;

wherein the agglomerator further comprises a mixing blade, wherein themixing blade is rotated at a blade rotation rate of about 500 rpm toabout 4000 rpm;

wherein exposing the agglomerated nutrient medium to drying conditionscomprises exposing the agglomerated nutrient medium to a predeterminedtemperature of about 60° C. to about 85° C.;

wherein the feed rate, the spray rate, the blade rotation rate, and thepredetermined temperature are selected to produce a dried agglomeratednutrient medium having a preselected mean water content of less than 5weight percent.

Embodiment G is the method of any one of the preceding Embodiments:

wherein introducing a nutrient component into the agglomeration chambercomprises introducing the nutrient component at a feed rate of about 50kg/hour to about 1000 kg/hour;

wherein introducing a agglomeration liquid into the agglomerationchamber comprises introducing the agglomeration liquid at a spray rateof about 15 kg/hour to about 300 kg/hour;

wherein the agglomerator further comprises a mixing blade, wherein themixing blade is rotated at a blade rotation rate of about 500 rpm toabout 4000 rpm;

wherein exposing the agglomerated nutrient medium to drying conditionscomprises exposing the agglomerated nutrient medium to a predeterminedtemperature of about 60° C. to about 85° C.;

wherein the feed rate, the spray rate, the blade rotation rate, and thepredetermined temperature are selected to produce a dried agglomeratednutrient medium having a gravity-packed density of 0.2 to 0.5 grams percubic centimeter.

Embodiment H is the method of any one of the preceding Embodiments:

wherein introducing a nutrient component into the agglomeration chambercomprises introducing the nutrient component at a feed rate of about 50kg/hour to about 1000 kg/hour;

wherein introducing a agglomeration liquid into the agglomerationchamber comprises introducing the agglomeration liquid at a spray rateof about 15 kg/hour to about 300 kg/hour;

wherein the agglomerator further comprises a mixing blade, wherein themixing blade is rotated at a blade rotation rate of about 500 rpm toabout 4000 rpm;

wherein exposing the agglomerated nutrient medium to drying conditionscomprises exposing the agglomerated nutrient medium to a predeterminedtemperature of about 60° C. to about 85° C.;

wherein the feed rate, the spray rate, the blade rotation rate, and thepredetermined temperature are selected to produce a dried agglomeratednutrient medium having particles of a first particle size distributionrange of about 105 microns to about 2000 microns.

Embodiment I is the method of any one of the preceding Embodiments,further comprising selecting a subpopulation of the particles, thesubpopulation having a second particle size distribution range that isnarrower than the first particle size distribution range, the secondparticle size distribution range being about 149 microns to about 1000microns.

Embodiment J is the method of any one of the preceding Embodiments,wherein one or more powdered nutrient is selected from the groupconsisting of a protein, a carbohydrate, a salt and a mixture of any twoor more of the foregoing powdered nutrients.

Embodiment K is the method of any one of the preceding Embodiments,further comprising the step of subjecting the dried agglomeratednutrient medium to a process that reduces the number of viablemicroorganisms in the dried agglomerated nutrient medium.

Embodiment L is the method of Embodiment K, wherein subjecting the driedagglomerated nutrient medium to a process that reduces the number ofviable microorganisms comprises exposing the dried agglomerated nutrientmedium to ionizing radiation or to ethylene oxide vapor.

Embodiment M is a composition comprising the flowable, driedagglomerated nutrient medium produced by the method of any one of thepreceding Embodiments.

Embodiment N is the composition of Embodiment M, wherein a specifiedmass of the dried agglomerated nutrient medium dissolves more rapidlythan an equal mass of the nutrient component from which the agglomeratednutrient medium was made.

Embodiment O is a kit comprising the composition of Embodiment M orEmbodiment N.

Embodiment P is the kit of Embodiment O, wherein the composition isdisposed in a package comprising a predetermined mass of the driedagglomerated nutrient medium, wherein the predetermined mass is selectedto be mixed with 9.9 mL, 10 mL, 90 mL, 99 mL, 100 mL, 225 mL, 1.0 L, or3.375 L of aqueous diluent to produce a reconstituted medium capable offacilitating the growth of a microorganism.

Embodiment Q is the kit of Embodiment O or Embodiment P, furthercomprising an article selected from the group consisting of a bag, abottle, and a sample acquisition device.

Embodiment R is the kit of any one of Embodiments O through Q, furthercomprising a selective agent and/or an indicator reagent.

Embodiment S is a thin film culture device, comprising:

a self-supporting waterproof substrate having first and second majorsurfaces;

an adhesive layer disposed on at least a portion of the first majorsurface;

a coating comprising the composition of Embodiment M or Embodiment Ndisposed on a least a portion of the adhesive layer; and

a dry, cold-water soluble gelling agent positioned for fluidic contactwith the nutrient medium.

Embodiment T is the thin film culture device of Embodiment S, furthercomprising a cover sheet coupled to a least a portion of the substrate,the cover sheet having a first side facing the first major surface.

Embodiment U is the thin film culture device of Embodiment T, whereinthe gelling agent is adhered to the first side of the cover sheet.

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention.

EXAMPLES Example 1. Production of Agglomerated Nutrient Medium ParticlesUsing a Schugi Agglomeration Process

A vertical Schugi FLEXOMIX FXD-100 mixer/agglomerator (BepexInternational LLC, Minneapolis, Minn.) is used for wet agglomeration. 25kilograms of dehydrated Buffered Peptone Water (“BPW”, ISO formulation,catalog number BPW010), obtained from 3M Company (St. Paul Minn.), isfed continuously via a powder dispenser at a feed rate of 300 kg/hr. Themixer/agglomerator is also fed continuously (via an array of 4atomization nozzles) with deionized water at a feed rate of 15 L/hr. Therotating knife blade is set to a speed of 4000 rpm. The moist granulatedpowder at the outlet of the mixer-granulator falls continuously undergravity into a fluidized bed dryer-matched with 3 compartments. Only thefirst zone is used to dry the wet-agglomerated product using air heatedto 60-65° C. The product is kept dry until the exhaust air reaches about45° C. The dried, agglomerated product is sieved to collect the powderbetween 20-140 mesh with a major portion (e.g., more than 40%) having anaffective diameter between 40-60 mesh (i.e., about 250 microns to about400 microns). The free-flow agglomerated BPW particles obtained usingthis process have characteristic properties that include agravity-packed density around 0.3-0.5 g/cm³ and a dissolution time ofless than 5 minutes in deionized water. The moisture content ofagglomerated BPW particles is less than 5%.

Example 2-4. Production of Agglomerated Nutrient Medium Particles Usinga Schugi Agglomeration Process

For Examples 2-4, the same procedure as described in Example 1 isfollowed with the exception that the operational conditions andinstrument settings (i.e. powder feed rate, liquid feed rate, bladerotating speed, drying temperature) are adjusted to the values shown inTable 1.

TABLE 1 Agglomeration parameters for Examples 1-4. Liquid Example Powderfeed feed rate Blade rotating Drying No. rate (kg/hr) (liter/hour) speed(rpm) temperature (° C.) 1 300 15 4000 60-65 2 300 22 4000 60-65 3 30030 4000 60-65 4 300 15 3000 60-65

Example 5-7. Production of Agglomerated Nutrient Medium Particles Usinga Schugi Agglomeration Process

A vertical Schugi FLEXOMIX FX-100 with N13 nozzle combo/Fluid bed FB-5(Bepex International LLC, Minneapolis, Minn.) is used for wetagglomeration. 25 kilograms of dehydrated Buffered Peptone Water (“BPW”,ISO formulation, catalog number BPW010), obtained from 3M Company (St.Paul Minn.), is fed continuously via a powder dispenser at a feed rateof 136 kg/hr. The FX100/FB5 is also fed continuously (via an array of 4atomization nozzles) with deionized water at a feed rate of 11.3 L/hr(example 5), 12.9 L hr (example 6), and 8.3 L/hr of 10% (w/w) of BPWsolution (Example 7). The rotating knife blade is set to a speed of 4000rpm. The moist granulated powder at the outlet of the mixer-granulatorfalls continuously under gravity into a fluidized bed dryer-matched with3 compartments. Only the first zone is used to dry the wet-agglomeratedproduct using air heated to below 80° C. The product is kept dry untilthe exhaust air reaches about 45° C. Table 2 shows the runningconditions for each example and Table 3 shows the particle analysisresulted from the process.

TABLE 2 Agglomeration parameters for Examples 5-7. Powder Liquid feedBlade Example Feeding feed rate rate rotating % Liquid No. liquid(kg/hr) (liter/hour) speed (rpm) addition 5 Water 136 11.3 4000 7.7 6Water 136 12.9 4000 8.6 7 10% 136 8.3 4000 5.7 BPW

TABLE 3 Analytical results for final product for Examples 5-7. Loss BulkExample Moisture density % Plus Mesh Size No. (%) (g/cm³) 40 60 80 100140 200 400 5 Water 136 1.0 7.7 15.9 23.9 49.5 73.2 92.5 6 Water 13631.5 57.1 74.2 81.5 89.9 95.5 98.9 7 10% 136 12.4 29.4 44.7 51.9 72.486.1 96.8 BPW

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. In the event that any inconsistency existsbetween the disclosure of the present application and the disclosure(s)of any document incorporated herein by reference, the disclosure of thepresent application shall govern. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

Various modifications may be made without departing from the spirit andscope of the invention. These and other embodiments are within the scopeof the following claims.

What is claimed is:
 1. A method of making a flowable, dried agglomeratednutrient medium, the method comprising: introducing a nutrient componentcomprising a powdered nutrient, and an agglomeration liquid, into anagglomerator comprising a flow-through-type agglomeration chamber and amixing blade; wherein the nutrient component facilitates the growth of amicroorganism; wherein the agglomeration liquid is introduced as anatomized spray; wherein introducing the nutrient component into theagglomeration chamber comprises introducing the nutrient component at afeed rate of about 50 kg/hour to about 1000 kg/hour; wherein introducingthe agglomeration liquid into the agglomeration chamber comprisesintroducing the agglomeration liquid at a spray rate about 15 kg/hour toabout 300 kg/hour; wet-massing the nutrient component with theagglomeration liquid in the agglomeration chamber for a period of timeto form agglomerated nutrient medium particles; exposing theagglomerated nutrient medium particles to drying conditions for a periodof time to form the dried, agglomerated nutrient medium; wherein themixing blade is rotated at a blade rotation rate of about 500 rpm toabout 4000 rpm; wherein exposing the agglomerated nutrient medium todrying conditions comprises exposing the agglomerated nutrient medium toa temperature of about 60° C. to about 85° C.; wherein the feed rate,the spray rate, the blade rotation rate, and the temperature areselected to produce a dried agglomerated nutrient medium havingparticles of a first particle size distribution range of about 105microns to about 2000 microns; and isolating a subpopulation of theparticles, the subpopulation having a second particle size distributionrange that is narrower than the first particle size distribution range,the second particle size distribution range being about 149 microns to1000 microns; wherein the powdered nutrient is capable of beingrehydrated to produce buffered peptone water, trypticase soy broth,lactose broth, UVM modified Listeria enrichment broth, buffered listeriaenrichment broth, or Demi-Fraser broth.
 2. The method of claim 1,wherein the agglomeration liquid consists of water.
 3. The method ofclaim 1, wherein the agglomeration liquid comprises a solvent having adissolved nutrient that facilitates the growth of a microorganism. 4.The method of claim 1, wherein introducing a nutrient componentcomprises introducing a substantially uniform mixture of two or morepowdered nutrients.
 5. The method of claim 1, wherein one or morepowdered nutrient is selected from the group consisting of a protein, acarbohydrate, a salt and a mixture of any two or more of the foregoingpowdered nutrients.
 6. The method of claim 1, further comprising thestep of subjecting the dried agglomerated nutrient medium to a processthat reduces the number of viable microorganisms in the driedagglomerated nutrient medium.
 7. The method of claim 6 whereinsubjecting the dried agglomerated nutrient medium to a process thatreduces the number of viable microorganisms comprises exposing the driedagglomerated nutrient medium to ionizing radiation or to ethylene oxidevapor.
 8. A method of making a thin film culture device, comprisingmaking a flowable, dried agglomerated nutrient medium according to themethod of claim 1; and disposing the flowable, dried agglomeratednutrient medium on an adhesive layer of a self-supporting waterproofsubstrate having first and second major surfaces and an adhesive layerthat is disposed on at least a portion of the first major surface suchthat the flowable, dried agglomerated nutrient medium is in fluidiccontact with a dry, cold-water gelling agent.
 9. The method of claim 8,wherein a cover sheet is coupled to at least a portion of theself-supporting waterproof substrate.
 10. The method of claim 9, whereinthe cover sheet has a first side facing the first major surface, andfurther wherein at least some of the dry, cold-water gelling agent isadhered to at least a portion of a first side of the cover sheet.